SUMMARY Phosphorylation of the Rb tumor suppressor (Rb) plays an important role in tumorigenesis. The molecules that control Rb phosphorylation in ?the Rb pathway? consisting of D-type cyclins, cyclin dependent kinases (CDKs) and CDK inhibitors (CDKIs) as well as Rb itself are consistently disrupted in most cancer types leading to excessive phosphorylation (hyper-phosphorylation) of the Rb protein. Hyperphosphorylation of Rb stimulates tumor growth by promoting proliferation, blocking apoptosis and increasing invasiveness. Recently inhibitors of kinases that phosphorylate Rb (CDK4/6) called palbociclib, ribociclib and abemaciclib have been approved by the FDA to treat a subset of breast cancer patients. These treatments are used in combination with hormonal or targeted therapies. Unfortunately resistance develops in response to CDK4/6 inhibitors that is due to the activation of the AKT/mTOR pathway. In this project we will test an alternate method of targeting Rb phosphorylation by the activation of phosphatase activity toward Rb. We predict that activation of phosphatase will result in the dephosphorylation of Rb without stimulation of the AKT/mTOR resistance pathway. These studies will include the evaluation of phosphatase activation in combination with hormonal or targeted therapies in three representative types of breast cancer cells (ER+,HER2+,or TNBC). Overall this project will elucidate the difference between inhibition of kinase activity and activation of phosphatase activity toward Rb phosphorylation with respect to key features of tumorigenesis: proliferation, apoptosis and invasion. These studies will yield useful information that could inform the development of future therapies that target Rb phosphorylation.